Chelating agents with lipophilic carriers

ABSTRACT

Compounds useful for associating with nanoparticle or microparticle emulsions to obtain magnetic resonance images permit control of the relaxivity of the signal and readily associate with the particulate components.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/765,299, now U.S.Pat. No. 7,279,150, filed 26 Jan. 2004 and is a continuation-in-part ofU.S. Ser. No. 10/351,463, now U.S. Pat. No. 7,255,875, filed 24 Jan.2003, which claims benefit from U.S. Ser. No. 60/351,390, filed 24 Jan.2002. This application also claims benefit of provisional application60/485,970, filed 9 Jul. 2003. The contents of these applications areincorporated herein by reference in their entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was supported in part by a grant from the U.S.Government. The U.S. Government has certain rights in this invention.

TECHNICAL FIELD

The invention is directed to chelating agents useful to support metalions employed in magnetic resonance imaging (MRI) where the chelate issupplied in a carrier which comprises lipophilic particles or droplets.More specifically, the invention is directed to chelating agentscoupled, optionally through a spacer, to phosphoglycerides.

BACKGROUND ART

The use of chelating agents of various types to entrap metal ions usefulin magnetic resonance imaging is well known. Generally, the chelatingagents contain a substantial number of unshared electron pairs ornegatively charged or potentially negatively charged species. Perhapsthe simplest among these is ethylenediaminetetraacetic acid (EDTA)commonly used as a water softener. However, many such agents are known,including, most notably, and commonly used, diethylene triaminepentaacetic acid (DTPA) and tetraazacyclododecanetetraacetic acid (DOTA)and their derivatives. U.S. Pat. Nos. 5,573,752 and 6,056,939,incorporated herein by reference, disclose particularly usefulderivatives of DOTA which are coupled to a benzyl or phenyl moietywherein the phenyl ring is substituted by isothiocyanate. Thisisothiocyanate provides a reactive group for coupling to variousadditional compounds. As described in these patents, the isothiocyanategroup can be used to couple the chelate to a targeting agent such as anantibody or fragment thereof.

There is an extensive literature on delivery vehicle compositions thathave been used to administer chelated metals for MRI. Some of thesecompositions do not contain targeting agents, though others do comprisesuch agents. For example, U.S. Pat. Nos. 5,690,907; 5,780,010;5,989,520; 5,958,371; and PCT publication WO 02/060524, the contents ofwhich are incorporated herein by reference, describe emulsions ofperfluorocarbon nanoparticles that are coupled to various targetingagents and to desired components, such as MRI imaging agents,radionuclides, and/or bioactive agents. Other compositions that havebeen used for targeted imaging include those disclosed in PCTpublications WO 99/58162; WO 00/35488; WO 00/35887; and WO 00/35492. Thecontents of these publications are also incorporated herein byreference.

The present invention in one embodiment is focused on improvements inthe contrast agents useful in magnetic resonance imaging; somebackground information on this technique is appropriate in understandingthe approach taken by applicants.

Magnetic resonance imaging (MRI) has become a useful tool for diagnosisand for research. The current technology relies on detecting the energyemitted when the hydrogen nuclei in the water contained in tissues andbody fluids returns to a ground state subsequent to excitation with aradio frequency. Observation of this phenomenon depends on imposing amagnetic field across the area to be observed, so that the distributionof hydrogen nuclear spins is statistically oriented in alignment withthe magnetic field, and then imposing an appropriate radio frequency.This results in an excited state in which this statistical alignment isdisrupted. The decay of the distribution to the ground state can then bemeasured as an emission of energy, the pattern of which can be detectedas an image.

While the above described process is theoretically possible, it turnsout that the relaxation rate of the relevant hydrogen nuclei, left totheir own devices, is too slow to generate detectable amounts of energy,as a practical matter. In order to remedy this, the area to be imaged issupplied with a contrast agent, generally a strongly paramagnetic metal,which effectively acts as a catalyst to accelerate the decay, thuspermitting sufficient energy to be emitted to create a detectable brightsignal. To put it succinctly, contrast agents decrease the relaxationtime and increase the reciprocal of the relaxation time—i.e., the“relaxivity” of the surrounding hydrogen nuclei.

Two types of relaxation times can be measured. T₁ is the time for themagnetic distribution to return to 63% of its original distributionlongitudinally with respect to the magnetic field and the relaxivity ρ₁,is its reciprocal. T₂ measures the time wherein 63% of the distributionreturns to the ground state transverse to the magnetic field. Itsreciprocal is the relaxivity index ρ₂. In general, the relaxation timesand relaxivities will vary with the strength of the magnetic field; thisis most pronounced in the case of the longitudinal component.

Thus, a desirable characteristic of any contrast agent is to provide thesignal with an enhanced relaxivity both for ρ₁ and ρ₂. The presentinvention provides such contrast agents.

It is also advantageous to facilitate the excretion of the paramagneticion, which may otherwise be toxic if it is retained in a subject. Thus,it would be advantageous to provide a mechanism for cleaving thechelated metal ion from the particles or from any lipid components thatmight result in cellular or liver uptake.

There is an extensive literature regarding contrast agents which arebased on chelated paramagnetic metals. For example, U.S. Pat. Nos.5,512,294 and 6,132,764 describe liposomal particles with metal chelateson their surfaces as MRI contrast agents. U.S. Pat. Nos. 5,064,636 and5,120,527 describe paramagnetic oil emulsions for MRI in thegastrointestinal tract. U.S. Pat. Nos. 5,614,170 and 5,571,498 describeemulsions that incorporate lipophilic gadolinium chelates, e.g.,gadolinium diethylenetriaminepentaacetic acid-bisoleate (Gd-DTPA-BOA) asblood pool contrast agents.

U.S. Pat. No. 5,804,164 describes water-soluble, lipophilic agents whichcomprise particularly designed chelating agents and paramagnetic metals.U.S. Pat. No. 6,010,682 and other members of the same patent familydescribe lipid soluble chelating contrast agents containing paramagneticmetals which are said to be able to be administered in the form ofliposomes, micelles or lipid emulsions.

Thus, in general, contrast agents may take the form of paramagneticmetals such as rare earth metals or iron mobilized in a form thatpermits substantial concentrations of the paramagnetic metal to bedelivered to the desired imaging area.

One method for providing useful concentrations of contrast agents hasbeen described by the present applicants in U.S. Pat. Nos. 5,780,010 and5,909,520. A nanoparticle is formed from an inert core surrounded by alipid/surfactant coating. The lipid/surfactant coating can then bemodified to couple the particle to a chelating agent containing aparamagnetic metal. In addition, the particle can be coupled to a ligandfor targeting to a specific site.

The present invention in one aspect provides an improvement in thedesign of contrast agents whereby the relaxivity of the signal can becontrolled, and excretion can be facilitated. The compounds of theinvention, however, are useful in other contexts as well, such asdelivering radionuclides to desired locations for imaging based onnuclear emissions.

DISCLOSURE OF THE INVENTION

The invention provides compounds which can readily be associated withcarriers of a variety of lipophilic delivery vehicles such as liposomes,fluorocarbon nanoparticles, oil droplets, and the like in a positionrelative to these delivery vehicles that provides for control ofrelaxivity of the signal and also provides, if desired, a mechanism forfacilitating excretion of the potentially toxic paramagnetic ion thatenhances the resonance image. In an alternative to chelation of aparamagnetic ion, a radioactive nuclide may be included; thedesirability of facilitating excretion of this nuclide is also apparent.The paramagnetic ion or radionuclide is provided in a chelate containedin compounds of the formula:

wherein Ch represents a chelating moiety;

-   -   m is 0-3;    -   R¹ is a non-interfering substituent;    -   1 is 0-2;    -   Z is S or O;    -   R² is H or alkyl (1-4 C);    -   n is 0 or 1; and    -   each R³ is independently an optionally substituted saturated or        unsaturated hydrocarbyl group containing at least 10 C.

The compounds of formula (1) may also comprise, associated with thechelating agent, at least one paramagnetic metal ion or a radionuclide.

In additional aspects, the invention is directed to compositionscomprising lipophilic delivery vehicles associated with the compounds offormula (1) and methods to obtain magnetic resonance or radionuclideimages using these compositions. In still other aspects, the inventionis directed to methods to prepare the compounds of formula (1) and tomethods to prepare the delivery vehicle compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the results of HPLC on Gd-MeO-DOTA-NCS andGd-MeO-DOTA-PE, respectively.

FIG. 2 shows the mass spectrum for Gd-MeO-DOTA-PE.

FIG. 3 shows a process flow chart for the preparation of Gd-MeO-DOTA-PE.

FIG. 4 shows the ρ₁ (relaxivity) value for various particulate chelatepreparations on a per ion basis.

FIG. 5 shows the ρ₁ relaxivity values for these particulate chelates ona per particle basis.

FIGS. 6A and 6B show the percent gadolinium retained in liver andspleen, respectively, in animals administered particulate chelates withand without cleavable triglycine linker.

MODES OF CARRYING OUT THE INVENTION

In general, the invention is directed to compounds of formula (1),including these compounds which comprise a paramagnetic metal ion or aradionuclide. In one embodiment, the invention does not includecompositions or compounds which comprise the specific structure setforth in Example 1.

The compounds of formula (1), when they include an appropriateparamagnetic ion, provide a conveniently prepared MRI contrast agentthat has at least two useful features. First, by virtue of its couplingto a phospholipid, it is readily associated with lipophilic deliveryvehicles such as liposomes, fluorocarbon nanoparticles, and the like.Second, because it may contain a spacer, the relaxivity of the signalcan be controlled by the distance imposed by the spacer from thesupporting delivery vehicles. An optional third advantage is that thespacer may provide a cleavage site which permits the contrast agent tobe dissociated from the particles and excreted once the image isobtained. The compounds of formula (1) are conveniently prepared fromisocyanate or isothiocyanate coupled to the benzene ring associated withthe chelating agent. Because of the reactivity of these groups, couplingcan be performed to a wide variety of spacers and phospholipids.

The chelating agents represented by Ch typically comprise at least two,and preferably a multiplicity of nitrogens spaced by alkylene groups andto which carboxylic acid-bearing moieties are coupled. Chelating agentsare characterized by comprising a multiplicity of unshared electronpairs or potential negative charges which serve to sequester the desiredmetal ion. Commonly employed chelating agents include porphyrins,ethylenediaminetetraacetic acid (EDTA),diethylenetriamine-N,N,N′,N″,N″-pentaacetate (DTPA),1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7 (ODDA),16-diacetate,N-2-(azol-1(2)-yl)ethyliminodiacetic acids,1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA),1,7,13-triaza-4,10,16-trioxacyclo-octadecane-N,N′,N″-triacetate(TTTA), tetraethylene glycols,1,5,9-triazacyclododecane-N,N′,N″,-tris(methylenephosphonic acid(DOTRP),N,N′,N″-trimethylammonium chloride (DOTMA) and analoguesthereof. A particularly preferred chelating agent in the compounds ofthe invention is DOTA.

The purpose of the chelating agent is, of course, to sequester thedesired paramagnetic metals or radionuclides. Suitable paramagneticmetals include a lanthanide element of atomic numbers 58-70 or atransition metal of atomic numbers 21-29, 42 or 44, i.e., for example,scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,copper, molybdenum, ruthenium, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, and ytterbium, most preferably Gd(III),Mn(II), iron, europium and/or dysprosium. Suitable radionuclides includethe radioactive forms of, for example, Sm, Ho, Y, Pm, Gd, La, Lu, Yb,Sc, Pr, Tc, Re, Ru, Rh, Pd, Pt, Cu, Au, Ga, In, Sn, and Pb.

The invention is not limited to compositions of these exemplaryradionuclides and paramagnetic ions; however, the foregoing lists arerepresentative.

The phosphoglyceride included in formula (1) is most convenientlyderived from naturally occurring lecithins, wherein the groupsrepresented by R³COO are fatty acids, such as oleic, palmitic, stearic,and the like. However, equally useful in the method of the invention arephosphoglycerides where each R³ is an optionally substituted hydrocarbylmoiety which may be saturated or unsaturated. The hydrocarbyl moietyshould contain at least 10C in order to confer sufficient lipophilicity;however, the carbons may be spaced apart by one or two heteroatomsselected from O, N or S. Suitable substituents include substituents thatcomprise aromatic moieties including heteroatom-containing aromaticmoieties, and/or the substituents may be halo, ═O, OR, SR, and NR₂wherein each R is independently an optionally substituted alkyl (1-6C).The hydrocarbyl moiety represented by R³ may be branched or straightchain and may comprise one or more cyclic portions. In general, each R³is simply of sufficient lipophilicity to provide a means for associationwith the lipophilic particulates or droplets that comprise the carrier.The skilled artisan can readily select embodiments for R³ which fulfillthis condition.

The spacer moiety noted in the formula may or may not be present. Thespacer may include a portion which has its origin in thephosphoglyceride itself—for example, in one important embodiment, thespacer may be or include the moiety CH₂CH₂ derived from aphosphodiglyceride which is a phosphatidyl ethanolamine, wherein the NR²shown in formula (1) is derived from a phosphatidyl ethanolamine.Preferred embodiments of R² include methyl, ethyl and H. In someembodiments, the spacer includes portions derived from peptides,pseudopeptides, polyalkylene glycols, such as polyethylene glycol, andthe like. (Pseudopeptides are polymers similar to peptides where thepeptide linkages have been replaced by isosteric linkages—i.e., whereinCONH linkages are replaced, for example, with CH₂NH, CH═CH, and thelike.) The length of the spacer may be chosen to control the relaxivityof the signal as described hereinbelow, and further may contain acleavage site which permits release of the chelate from the carrierparticle.

The “non-interfering substituent” R¹ on the benzene ring in formula (1)is any substituent, such as alkyl (1-6C), halo, alkoxy (1-6C), and thelike, which does not interfere with the coupling of the chelating agentto the remainder of the molecule, or with the ability of the chelatingagent to entrap a suitable metal ion, or with the use of thecompositions containing the compound of formula (1) in imaging. Methoxyis preferred. It is understood that a variety of substitutions may bepresent on the benzene ring without interference with the essentialfeatures of the compound. Any substituent found to detract significantlyfrom the performance of the compound of formula (1) in chelating metalsor in participating in imaging is not included within the scope of theinvention. Suitable substituents include OR, NR₂, SR, CN, NO₂, SO₃H, andR where R is alkyl or alkenyl optionally substituted by, e.g., halo, ═O,and the like and optionally containing a heteroatom, such as O, S or N.

In general, the compounds of formula (1) are synthesized from a compoundof the formula:

wherein m, 1 and R¹ are defined as above with a compound of the formula:

wherein R², n and R³ are defined as above. Reactions of this type arefacile and conditions for the conduct of such reactions are well knownin the art. Typically, the reaction is conducted in an aprotic solventin the presence of a weak base.

The compounds of formula (1), typically associated with the metal, areincluded in compositions which contain lipophilic delivery vehicles.“Delivery vehicles” are particulate carriers that are, at least on theirsurface, lipophilic and which are suspended in a hydrophilic or aqueousmedium. These vehicles are microparticles or nanoparticles, and may haveaverage diameters in the range of 10 nm-100 μm, preferably 50 nm-50 μm.However, for in vivo use, particles having diameters in the range of50-500 nm, preferably 50-300 nm are preferred. The particles may be of avariety of compositions, including such well known vehicles asliposomes, which may be of various sizes and may be unilamellar ormultilamellar, micelles, oil droplets, lipoproteins, such as HDL, LDL,IDL, VLDL, chylomicrons, fluorocarbon nanoparticles, microbubbles ornanobubbles, or any of a wide variety of particles in the abovementioned size range that are lipophilic at least at their surface, asfurther described below. Thus, the surface of these nanoparticles willcomprise lipids or surfactants or both.

The compounds of formula (1), when associated with a paramagnetic ionand the lipophilic particles contained in a carrier system are useful inobtaining magnetic resonance images. The vehicles in the delivery systemmay further comprise other useful components such as targeting agents tocarry the contrast agent to the desired tissue or organ and mayoptionally contain therapeutic or other biologically active agents. Insome embodiments, these vehicles may also comprise other imaging agentssuch as radionuclides, or, more commonly, include the radionuclides, inthe alternative, in the chelate.

Targeting agents typically may comprise antibodies or immunospecificfragments thereof, ligands for receptors present on the desired targetor tissue, molecules designed specifically to target cellular componentssuch as those designed based on cyclic RGD peptides designed to targetintegrins and the like. The lipophilic particles themselves may includereactive groups that can be coupled to targeting agents.

Lipid/surfactant components of the delivery vehicles can be coupled tothese reactive groups through functionalities contained in thelipid/surfactant component. For example, phosphatidylethanolamine may becoupled through its amino group directly to a desired moiety, or may becoupled to a linker such as a short peptide which may provide carboxyl,amino, or sulfhydryl groups as described below. Alternatively, standardlinking agents such a maleimides may be used. A variety of methods maybe used to associate the targeting ligand and the ancillary substancesto the nanoparticles; these strategies may include the use of spacergroups such as polyethylene glycol or peptides, for example.

For coupling by covalently binding the targeting ligand or other organicmoiety to the components of the outer layer, various types of bonds andlinking agents may be employed. Typical methods for forming suchcoupling include formation of amides with the use of carbodiamides, orformation of sulfide linkages through the use of unsaturated componentssuch as maleimide. Other coupling agents include, for example,glutaraldehyde, propanedial or butanedial, 2-iminothiolanehydrochloride, bifunctional N-hydroxysuccinimide esters such asdisuccinimidyl suberate, disuccinimidyl tartrate,bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, heterobifunctionalreagents such as N-(5-azido-2-nitrobenzoyloxy)succinimide, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and succinimidyl4-(p-maleimidophenyl)butyrate, homobifunctional reagents such as1,5-difluoro-2,4-dinitrobenzene,4,4′-difluoro-3,3′-dinitrodiphenylsulfone,4,4′-diisothiocyano-2,2′-disulfonic acid stilbene,p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenylester), 4,4′-dithiobisphenylazide, erythritolbiscarbonate andbifunctional imidoesters such as dimethyl adipimidate hydrochloride,dimethyl suberimidate, dimethyl 3,3′-dithiobispropionimidatehydrochloride and the like. Linkage can also be accomplished byacylation, sulfonation, reductive amination, and the like. Amultiplicity of ways to couple, covalently, a desired ligand to one ormore components of the outer layer is well known in the art. The liganditself may be included in the surfactant layer if its properties aresuitable. For example, if the ligand contains a highly lipophilicportion, it may itself be embedded in the lipid/surfactant coating.Further, if the ligand is capable of direct adsorption to the coating,this too will effect its coupling. For example, nucleic acids, becauseof their negative charge, adsorb directly to cationic surfactants.

The targeting ligand or antibody may bind directly to the nanoparticle,i.e., the ligand or antibody is associated with the nanoparticle itself,as described above. Alternatively, indirect binding such as thateffected through biotin/avidin may be employed. Typically, inbiotin/avidin mediated targeting, the ligand or antibody is coupled notto the emulsion, but rather coupled, in biotinylated form, to thetargeted tissue.

Ancillary agents that may be coupled to the nanoparticles throughentrapment in the coating layer include radionuclides, instead of, or inaddition to, the paramagnetic ion. Radionuclides may be eithertherapeutic or diagnostic; diagnostic imaging using such nuclides iswell known and by targeting radionuclides to undesired tissue atherapeutic benefit may be realized as well. Typical diagnosticradionuclides include ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga,and therapeutic nuclides include ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir. The nuclide can beprovided to a preformed emulsion in a variety of ways. For example,⁹⁹Tc-pertechnate may be mixed with an excess of stannous chloride andincorporated into the preformed emulsion of nanoparticles. Stannousoxinate can be substituted for stannous chloride. In addition,commercially available kits, such as the HM-PAO (exametazine) kitmarketed as Ceretek® by Nycomed Amersham can be used. Means to attachvarious radioligands to the nanoparticles of the invention areunderstood in the art. As stated above, the radionuclide may not be anancillary material, but may instead occupy the chelating agent in lieuof the paramagnetic ion when the composition is to be used solely fordiagnostic or therapeutic purposes based on the radionuclide.

Other ancillary agents include fluorophores such as fluorescein, dansyl,quantum dots, and the like.

Included in the lipophilic carrier vehicle as ancillary agents, in someembodiments of the invention, are biologically active agents. Thesebiologically active agents can be of a wide variety, including proteins,nucleic acids, pharmaceuticals, and the like. Thus, included amongsuitable pharmaceuticals are antineoplastic agents, hormones,analgesics, anesthetics, neuromuscular blockers, antimicrobials orantiparasitic agents, antiviral agents, interferons, antidiabetics,antihistamines, antitussives, anticoagulants, and the like.

In all of the foregoing cases, whether the associated moiety is atargeting ligand for a tissue or organ or is an ancillary agent, thedefined moiety may be non-covalently associated with the lipophilicvehicle, may be directly coupled to the components of the vehicle, ormay be coupled to said components through spacer moieties.

A multiplicity of vehicles may be used in the compositions of theinvention, for example, liposomal particles. The literature describingvarious types of liposomes is vast and well known to practitioners. Asthe liposomes themselves are comprised of lipid moieties, theabove-described lipids and surfactants are applicable in the descriptionof moieties contained in the liposomes themselves. These lipophiliccomponents can be used to couple to the chelating agent in a mannersimilar to that described above with respect to the coating on thenanoparticles having an inert core. Micelles are composed of similarmaterials, and this approach to coupling desired materials, and inparticular, the chelating agents applies to them as well. Solid forms oflipids may also be used.

In another example, proteins or other polymers can be used to form theparticulate carrier. These materials can form an inert core to which alipophilic coating is applied, or the chelating agent can be coupleddirectly to the polymeric material through techniques employed, forexample, in binding affinity reagents to particulate solid supports.Thus, for example, particles formed from proteins can be coupled totether molecules containing carboxylic acid and/or amino groups throughdehydration reactions mediated, for example, by carbodiimides.Sulfur-containing proteins can be coupled through maleimide linkages toother organic molecules which contain tethers to which the chelatingagent is bound. Depending on the nature of the particulate carrier, themethod of coupling so that an offset is obtained between the dentateportion of the chelating agent and the surface of the particle will beapparent to the ordinarily skilled practitioner.

In still another example, PCT publication WO95/03829 describes oilemulsions where the drug is dispersed or solubilized inside an oildroplet and the oil droplet is targeted to a specific location by meansof a ligand. U.S. Pat. No. 5,542,935 describes site-specific drugdelivery using gas-filled perfluorocarbon microspheres. The drugdelivery is accomplished by permitting the microspheres to home to thetarget and then effecting their rupture. Low boiling perfluoro compoundsare used to form the particles so that the gas bubbles can form.

One important embodiment comprises emulsions wherein the nanoparticlesare based on high boiling perfluorocarbon liquids such as thosedescribed in U.S. Pat. No. 5,958,371 referenced above. The liquidemulsion contains nanoparticles comprised of relatively high boilingperfluorocarbons surrounded by a coating which is composed of a lipidand/or surfactant. The surrounding coating is able to couple directly toa targeting moiety or can entrap an intermediate component which iscovalently coupled to the targeting moiety, optionally through a linker,or may contain a non-specific coupling agent such as biotin.Alternatively, the coating may be cationic so that negatively chargedtargeting agents such as nucleic acids, in general or aptamers, inparticular, can be adsorbed to the surface.

One useful emulsion is a nanoparticulate system containing a highboiling perfluorocarbon as a core and an outer coating that is alipid/surfactant mixture which provides a vehicle for binding amultiplicity of copies of one or more desired components to thenanoparticle. The construction of the basic particles and the formationof emulsions containing them, regardless of the components bound to theouter surface is described in the above-cited patents to the presentapplicants, U.S. Pat. Nos. 5,690,907 and 5,780,010; and patents issuedon daughter applications U.S. Pat. Nos. 5,989,520 and 5,958,371 andincorporated herein by reference.

The high boiling fluorochemical liquid is such that the boiling point ishigher than that of body temperature—i.e., 37° C. Thus, fluorochemicalliquids which have boiling points at least 30° C. are preferred, morepreferably 37° C., more preferably above 50° C., and most preferablyabove about 90° C. The “fluorochemical liquids” useful in the inventioninclude straight and branched chain and cyclic perfluorocarbonsincluding perfluorinated compounds which have other functional groups.“Perfluorinated compounds” includes compounds that are not pureperfluorocarbons but rather wherein other halo groups may be present.These include perfluorooctylbromide, and perfluorodichlorooctane, forexample.

Useful perfluorocarbon emulsions are disclosed in U.S. Pat. Nos.4,927,623, 5,077,036, 5,114,703, 5,171,755, 5,304,325, 5,350,571,5,393,524, and 5,403,575, which are incorporated herein by reference,and include those in which the perfluorocarbon compound isperfluorodecalin, perfluorooctane, perfluorodichlorooctane,perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane,perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine,perfluortributylamine, perfluorodimethylcyclohexane,perfluorotrimethylcyclohexane, perfluorodicyclohexyl ether,perfluoro-n-butyltetrahydrofuran, and compounds that are structurallysimilar to these compounds and are partially or fully halogenated(including at least some fluorine substituents) or partially or fullyfluorinated including perfluoroalkylated ether, polyether or crownether.

It will be noted, that in addition to high boiling halo carbons, theparticles useful in the compositions of the invention may containmicrobubbles or nanobubbles. Thus, lower boiling components of theparticles may be employed such that at temperatures in vivo effectvaporization.

In addition, lipoproteins and chylomicrons may also be used. Varioustypes of lipoprotein are well known and include, for example, LDL, HDL,and VLDL.

In one embodiment, lipid/surfactant coated nanoparticles may be formedby microfluidizing a mixture of a fluorocarbon lipid which forms thecore and a lipid/surfactant mixture which forms the outer layer insuspension in aqueous medium to form an emulsion. In this procedure, thelipid/surfactants may already be coupled to additional ligands when theyare coated onto the nanoparticles, or may simply contain reactive groupsfor subsequent coupling. Alternatively, the components to be included inthe lipid/surfactant layer may simply be solubilized in the layer byvirtue of the solubility characteristics of the ancillary material.Sonication or other techniques may be required to obtain a suspension ofthe lipid/surfactant in the aqueous medium. Typically, at least one ofthe materials in the lipid/surfactant outer layer comprises a linker orfunctional group which is useful to bind an additional desired componentor the component may already be coupled to the material at the time theemulsion is prepared.

The lipid/surfactants used to form an outer coating on the deliveryvehicles (that will contain the coupled ligand or entrap reagents forbinding desired components to the surface) include natural or syntheticphospholipids, fatty acids, cholesterols, lysolipids, sphingomyelins,and the like, including lipid conjugated polyethylene glycol. Variouscommercial anionic, cationic, and nonionic surfactants can also beemployed, including Tweens, Spans, Tritons, and the like. Somesurfactants are themselves fluorinated, such as perfluorinated alkanoicacids such as perfluorohexanoic and perfluorooctanoic acids,perfluorinated alkyl sulfonamide, alkylene quaternary ammonium salts andthe like. In addition, perfluorinated alcohol phosphate esters can beemployed. Cationic lipids included in the outer layer may beadvantageous in entrapping ligands such as nucleic acids, in particularaptamers. Typical cationic lipids may include DOTMA,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP,1,2-dioleoyloxy-3-(trimethylammonio)propane; DOTB,1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol,1,2-diacyl-3-trimethylammonium-propane;1,2-diacyl-3-dimethylammonium-propane; 1,2-diacyl-sn-glycerol-3-ethylphosphocholine; and 3β-[N′,N′-dimethylaminoethane)-carbamol]cholesterol-HCl.

In some embodiments, included in the lipid/surfactant at the surface arecomponents with reactive groups that can be used to couple a targetingligand or antibody and/or the ancillary substance useful for imaging ortherapy.

Use of the Compositions in Magnetic Resonance Imaging

When used in magnetic resonance imaging, the compositions of theinvention typically contain a paramagnetic ion within the chelatingstructure. In such applications, the inclusion of a spacer isparticularly advantageous.

As set forth above, the function of the spacer is two-fold: first, bycontrolling the distance of the chelating agent and thereby theparamagnetic ion from the particles, the exposure of the paramagneticion to the hydrogen in the aqueous surroundings of the particles iscontrolled and thereby the relaxivity of the signal can be adjusted.Second, the spacer may include a cleavable group, thereby expediting theexcretion of the chelated metal ion when its imaging function has beenserved.

Turning first to the effect on relaxivity, to maximize the relaxivitiesobtainable, the dimensions of the spacer are such that the paramagneticion is offset from the surface of the particle at a distance,preferably, of at least 5 or 10 Å. Preferably the average distance atwhich the paramagnetic ion is found from the surface is between about5-100 Å, preferably about 10-50 Å, and more preferably about 10-20 Å.

As used herein, the “surface” of the vehicle means the outer limit ofthe material comprising the particle at the location at which thechelator is coupled. Overall, the mean diameter of the particle itselfis compared to the mean distance from the center where the paramagneticions reside. This should be at least a 5 Å difference preferably atleast 10 Å.

The degree of offset can also be defined in terms of the resultantimpact on the relaxivity imparted by the offset. The imparted relaxivityis dependent on the strength of the magnetic field; the relaxivity on aper particle basis is, of course, determined in part by the number ofparamagnetic ions associated with the particle itself. At thearbitrarily chosen magnetic field strength of 0.47 T, the offset will besufficient to enhance the relaxivity on a per ion basis at least 1.2fold, preferably 1.5 fold, and more preferably 2.5 fold or 10 fold forρ₁ and in similar amounts for ρ₂. At the arbitrarily chosen magneticfield of 1.5 T, the offsets will enhance these relaxivities by similarfactors. At 4.7 T, preferably the enhancement of ρ₁ is at least 1.5fold, preferably 2 fold and the enhancement of ρ₂ is at least two foldand preferably three fold, again, on a per ion basis. In terms of unitsof relaxivity per se, the offset is such that the value for ρ₁ in(s*mM)⁻¹ at 0.47 T is at least 20, and preferably 25, more preferably30; at 1.5 T, these values would be at least 20, and preferably 30, andat 4.7 T, at least 10, and preferably 14. For ρ₂, the correspondingvalues at 0.47 T would be at least 20, preferably 30, and morepreferably 35; at 1.5 T, at least 20, preferably 30; and at 4.7 T, atleast 20, more preferably 40, and most preferably 60.

By appropriately coupling the chelating agents, substantial numbers ofchelators and paramagnetic ions can be coupled to the particles. For thechelator containing a paramagnetic ion, typically, the particles containat least 2,000 copies, typically at least 5,000, more typically at least10,000 or 100,000 or 500,000. For targeting agents, only one or two, orseveral or more copies may be included. Variable numbers of drugmolecules may be contained.

As applicants are able to apply to the vehicles of the composition amultiplicity of chelators containing paramagnetic ions, considerablyhigher relaxivities can be obtained on a per particle basis. The foldincrease in ρ₁ and ρ₂ on a per particle basis is, of course, similar tothat with respect to the fold increase on a per ion basis. Applicants,however, have been able to achieve values of ρ₁ in units of (s*mM)⁻¹ ona per particle basis at 0.47 T, of at least 1.8×10⁶, preferably 2.0×10⁶,and more preferably 2.5×10⁶. At 1.5 T, these values are similar and at4.7 T, relaxivity values for ρ₁ are at least 8×10⁵, preferably 1×10⁶,more preferably 1.1×10⁶.

For ρ₂ at 0.47 T, the relaxivity is preferably at least 2×10⁶, morepreferably 2.5×10⁶, and more preferably 3×10⁶ in these units. At 1.5 T,the values for ρ₂ are at least 1.6×10⁶, preferably 2.5×10⁶, and morepreferably 3×10⁶. At 4.7 T, ρ₂ is at least 3×10⁶, more preferably 4×10⁶,and more preferably 5×10⁶.

The offsetting is accomplished by spacing the dentate portion of thechelate through the spacer to the surface of the vehicle, as thephosphoglyceride associates with the lipophilic material at the surface.

Cleavable Spacers

In a second advantage of use of spacers, the spacer may be cleavable sothat the paramagnetic ion or radionuclide ion chelate can be dissociatedfrom the particle or from lipids that compose part of the vehicle. Itmay be desirable to enhance excretion by liberating the chelate in ahydrophilic status to promote such excretion. Accordingly, the spacermay contain one or more cleavage sites that either are activatedexternally, for example, by photoactivation, or which are continuouslyaccessed by enzymes present in the cells or bloodstream. Examples of theformer include specific linkages that are photoactivated, or cleaved byultrasound, as is understood in the art. After imaging or therapy hasbeen completed, the nanoparticles are subjected to electromagneticenergy or ultrasound as appropriate to effect cleavage. In the secondinstance, the spacer may be, or may include, peptides containing aminoacid sequences that are susceptible to cleavage by circulating proteasesor may include polysaccharides, themselves susceptible to such cleavage.Any combination of such cleavage sites may be included. Thesusceptibility of the spacer or tether to cleavage thus enhancesexcretion and diminishes potential toxicity of the paramagnetic ion.

If continuous degradation is employed, the rate may be modulated byselecting spacers according to the available enzymatic activities and bysupplying a desired number of cleavage sites. However, it is well knownthat any peptide circulating in the bloodstream is ultimately destroyeddue to circulating proteases; similarly, polysaccharides are subject tocleavage by endogenous enzymes.

Methods of Preparation

The precise process for preparation of the compositions of the inventionis variable, and depends on the nature of the particulate vehicle andthe choice of spacer molecules, when present. As described above, solidparticles which contain reactive groups can be coupled directly to thespacer; lipid-based particles such as oil emulsions, solid lipids,liposomes, fluorocarbon nanoparticles and the like, can includelipophilic materials containing reactive groups which may covalently,then, be coupled to linking moieties which bear the dentate portion ofthe chelating agent. In one particularly preferred embodiment, theprocess involves mixing a liquid fluorocarbon compound that forms thecore of a nanoparticle and the components of a lipid/surfactant coatingfor that particle in an aqueous suspension, microfluidizing, and, ifdesired, harvesting and sizing the particles. The components to becoupled can be included in the original mixture by virtue of theirinitial coupling to one or more components of the lipid/surfactantcoating, or the coupling to additional moieties can be conducted afterthe particles are formed.

Kits

The emulsions of the invention may be prepared and used directly in themethods of the invention, or the components of the emulsions may besupplied in the form of kits. The kits may comprise the pre-preparedtargeted composition containing all of the desired ancillary materialsin buffer or in lyophilized form. Alternatively, the kits may include aform of the emulsion which lacks the compound of formula (1) and/or atargeting agent which is supplied separately. If the targeting agent isto be directly bound, the emulsion will contain a reactive group, suchas a maleimide group, which, when the emulsion is mixed with thetargeting agent, effects the binding of the targeting agent to theemulsion itself. A separate container may also provide additionalreagents useful in effecting the coupling. Alternatively, the emulsionmay contain reactive groups which bind to linkers coupled to the desiredcomponent to be supplied separately which itself contains a reactivegroup. A wide variety of approaches to constructing an appropriate kitmay be envisioned. Individual components which make up the ultimateemulsion may thus be supplied in separate containers, or the kit maysimply contain reagents for combination with other materials which areprovided separately from the kit itself.

A non-exhaustive list of combinations might include: emulsionpreparations that contain, in their lipid-surfactant layer, an ancillarycomponent such as a fluorophore or chelating agent and reactive moietiesfor coupling to the targeting agent; the converse where the emulsion iscoupled to targeting agent and contains reactive groups for coupling toan ancillary material; emulsions which contain both targeting agent anda chelating agent but wherein the metal to be chelated is eithersupplied in the kit or independently provided by the user; preparationsof the nanoparticles comprising the surfactant/lipid layer where thematerials in the lipid layer contain different reactive groups, one setof reactive groups for a targeting agent and another set of reactivegroups for an ancillary agent; preparation of emulsions containing anyof the foregoing combinations where the reactive groups are supplied bya linking agent.

Applications

The emulsions and kits for their preparation are useful in the methodsof the invention which include imaging of tissues and providingtherapeutic agents.

The magnetic resonance imaging contrast agents of the present inventionmay be used in a similar manner as other MRI agents as described in U.S.Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt, et al., Magn.Reson. Med. (1986) 3:808; Runge, et al., Radiology (1988) 166:835; andBousquet, et al., Radiology (1988) 166:693. Other agents that may beemployed are those set forth in U.S. patent publication 2002/0127182which are pH sensitive and can change the contrast properties dependenton pulse. Generally, sterile aqueous solutions of the contrast agentsare administered to a patient intravenously in dosages ranging from 0.01to 1.0 mmoles per kg body weight.

Usually, the diagnostic compositions for radionuclide imaging areadministered by intravenous injection, usually in saline solution, at adose of 1 to 100 mCi per 70 kg body weight, or preferably at a dose of 5to 50 mCi. Imaging is performed using known procedures.

The therapeutic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 0.01 to 5 mCi per kgbody weight, or preferably at a dose of 0.1 to 4 mCi per kg body weight.For comparable, i.e., analogous therapeutic radiopharmaceuticals,current clinical practice sets dosage ranges from 0.3 to 0.4 mCi/kg forZevalin™ to 1-2 mCi/kg for OctreoTher™, a labeled somatostatin peptide.For such therapeutic radiopharmaceuticals, there is a balance betweentumor cell kill vs. normal organ toxicity, especially radiationnephritis. At these levels, the balance generally favors the tumor celleffect. These dosages are higher than corresponding imaging isotopes.

When the compositions of the invention contain targeted deliveryvehicles, suitable targets include any tissue of interest, includingtumor tissue, atherosclerotic plaques, blood clots, and the like. Thechoice of targeting agent will, of course, depend on the nature of thetarget itself. For example, to target atherosclerotic plaques or bloodclots, antifibrin antibodies are appropriate as are peptidomimetics thatinteract with α_(v)β₃ receptors. Suitable targeting agents for tumorsmay include antibodies prepared against tumor associated antigens orprepared with respect to the organ hosting the tumor. Imaging ofparticular organs would employ targeting agents that interact withreceptors or other characteristic moieties associated with the targetitself.

The following examples are intended to illustrate but not to limit theinvention.

PREPARATION A Nanoparticle Preparation

Paramagnetic nanoparticles were produced in a modification of theprocedure described by Lanza, G, et al., Circulation (1996)94:3334-3340. Briefly, the emulsions comprised 40% (v/v)perfluorooctylbromide (PFOB; MMM, St. Paul, Minn.), 2% (w/v) saffloweroil, 2% (w/v) of a surfactant co-mixture, 1.7% (w/v) glycerin and waterrepresenting the balance. The surfactant co-mixture included 63 mole %lecithin (Avanti Polar Lipids, Inc., Alabaster, Ala.), 15 mole %cholesterol (Sigma Chemical Co., St. Louis, Mo.), 2 mole %dipalmitoyl-phosphatidylethanolamine (Avanti Polar Lipids, Inc.,Alabaster, Ala.), and 20 mole % of the paramagnetic lipophilic chelate.The lipophilic chelate was either gadoliniumdiethylene-triamine-pentaacetic acid-bis-oleate (Gd-DTPA-BOA; GatewayChemical Technologies, St. Louis, Mo.) or DTPA-phosphatidylethanolamine(DTPA-PE; Gateway Chemical Technologies, St. Louis, Mo.). The surfactantcomponents were dissolved in chloroform, evaporated under reducedpressure, dried in a 50° C. vacuum oven overnight and dispersed intowater by sonication. The suspension was pre-emulsified in a blender withPFOB, safflower oil and distilled deionized water for 30 to 60 secondsand then emulsified in a M110S Microfluidics emulsifier (Microfluidics,Newton, Mass.) at 20,000 PSI for four minutes. The completed formulationwas placed in crimp sealed vials and blanketed with nitrogen. Particlesizes were determined in triplicate at 37° C. with a laser lightscattering submicron particle sizer (Malvern Instruments, Malvern,Worcestershire, UK).

EXAMPLE 1 Preparation of a Compound of Formula (1)

Phosphoethanolamine diglyceride (PE) is first coupled to t-boc protectedtriglycine. Standard coupling techniques, such as forming the activatedester of the free acid of the t-boc-triglycine using diisopropylcarbodiimide (or an equivalent thereof) with either N-hydroxysuccinimide (NHS) or hydroxybenzotriazole (HBT) are employed and thet-boc-triglycine-PE is purified.

Treatment of the t-boc-triglycine-PE with trifluoroacetic acid yieldstriglycine-PE, which is then reacted with excess DOTA-NCS in DMF/CHCl₃at 50° C. for 8 hours. The final product is isolated by removing thesolvent, followed by rinsing the remaining solid with excess water, toremove excess solvent and any un-reacted or hydrolyzed DOTA-NCS.

It will be noted that the triglycine spacer is a cleavable linker as asubstrate for proteases. Alternatively, instead of the triglycinespacer, a similar construct was prepared using caproylamine-PE, which iscommercially available from Avanti Polar Lipids. This material isreacted with DOTA-NCS in an analogous manner to that set forth withrespect to the glycine spacer described above.

EXAMPLE 2 Introduction of Gadolinium Ion

Gadolinium ion may be introduced into the chelate either by initiallymetalating DOTA-NCS or metalating the compound of formula (1) aftersynthesis.

Premetalation of MeO-DOTA-NCS was carried out in aqueous GdCl₃. Thereaction mixture was lyophilized to dryness and used without furtherpurification prior to conjugation with PE or triglycyl-PE. As the saltscarried onto the final conjugation negatively affect the couplingchemistry, they were removed by aqueous rinses of the driedMeODOTA-Gd-PE reaction mixture.

Gd₂O₃ may be used in place of GdCl₃ to produce a “salt free” metalcomplex, by boiling the solution containing Gd₂O₃ for an extended periodof time in MeOH/chloroform.

Postmetalation of conjugated MeO-DOTA-PE is carried out with GdCl₃ in achloroform methanol mixture with boiling.

Gd-MeO-DOTA was characterized by HPLC. LC conditions were: Zorbax CB C8column, 25% acetonitrile, 0.2% TFA isocratic elution, detection at 260nm. Uncomplexed MeO-DOTA-NCS elutes at 3.8-4.0 min. MeO-DOTA-PE wascharacterized by LC and MS. The HPLC conditions for the characterizationof Gd-MeODOTA-PE were: Astec, Pholipidec column; solvent system: solventA (80% CHCl3, 19% MeOH, 1% NH4OH) and solvent B (MeOH), gradientprofile: 0-10 min 100-75% A, 0-25% B; 10-15 min 25-100% B, hold at 100%B from 15-20 min, 100-0% B from 20-22 min, hold at 100% for 5 min. ELSDand UV detectors were employed.

The HPLC results for Gd-MeO-DOTA-NCS and for the corresponding conjugateGd-MeO-DOT-PE are shown in FIGS. 1A and 1B, respectively. The massspectrum of the resulting Gd-MeO-DOTA-PE is shown in FIG. 2.

EXAMPLE 3 Synthesis of Conjugate for Animal Studies

A. Preparation of GdMeODOTA-NCS

MeO-DOTA-NCS (Dow Chemical) (18.71 g, 33 mmol) was dissolved indeionized water (500 mL). A pH probe was placed into the solution andwhile being stirred, the pH of the solution was adjusted to ˜6 with theaddition of NaOH (50%). In a separate flask, GdCl₃×6H₂O (Ohduch) (18.37g) was dissolved in 100 mL of DI water. The Gd solution was carefullyadded to the stirring solution of 3 in 5 mL aliquots. After eachaddition, the pH was measured and adjusted PRN to a pH of 6-7 with theaddition of sodium hydroxide (50%). The solution was lyophilized todryness. This process produced 43.9 g of a faintly, green powder with anoverall purity of 92%.

B. Preparation of Gd-MeO-DOTA-PE

A 2 L, 3-neck round-bottom flask was charged with Gd-MeO-DOTA-NCSprepared in paragraph A. (43.9 g, mmol); PE (15 g, 22 mmol); DMF (500mL); Et3N (4.57 mL); and CHCl₃ (300 mL). PE was supplied as 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine obtained from Avanti.The mixture was heated for 8 hours at 50° C. The reaction was monitoredby HPLC. The solvents were removed in vacuo, and the resulting solid wassuspended in water (˜100 mL), then filtered over a bed of Celpurefiltering agent (8-10 cm thick), using a coarse fritted funnel. Thesolids were rinsed with copious amount of water (1 L-1.5 L). After themajority of the water had been removed from the solid cake layer, thesolids were rinsed with CHCl₃:MeOH (3:1) (total volume of ˜1 L-1.5 L).The organic filtrate solution was dried over sodium sulfate. The mixturewas filtered and dried in vacuo, leaving 20 g of light beige, glassy,solid. The overall purity was 90% based on LC.

A process flow chart for the preparation of the final product is shownin FIG. 3.

EXAMPLE 4 Effect of Spacer Length on Relaxivity

In this example, an embodiment of the invention employing DTPA as thechelator (Ch), gadolinium as the paramagnetic ion, ultimately linked tophosphatidyl ethanolamine was used to indicate the effect of spacerlength on relaxivity. Although the Gd-DTPA-PE itself does not fallwithin the scope of the compounds of the invention, the data in thisexample illustrate the effect of the spacing of the chelating agent fromthe particles in the composition on relaxivity. DTPA-PE can be purchasedfrom Gateway Chemical Technologies, St. Louis, Missouri. It was comparedwith the relaxivity generated by gadoliniumdiethylenetriaminepentaacetic acid-bisoleate (Gd-DTPA-BOA) which canalso be purchased from Gateway.

The nanoparticles were prepared as described in Preparation A and thechelates purchased from Gateway incorporated as there described.Gadolinium chloride was added in excess proportions as opposed toemulsification step to the nanoparticles formulated with DTPA-PE.Unbound gadolinium was removed by dialysis against distilled deionizedwater (300,000 mw cutoff, Spectrum Laboratories, Rancho Dominguez,Calif.). Gd-DTPA-BOA had been incorporated as the complete compound asdescribed. Both compositions were tested for free Gd³⁺using the arsenazoIII reaction and showed no sign of unbound gadolinium.

The Gd-DTPA-BOA and Gd-DTPA-PE nanoparticles had the followingcharacteristics:

TABLE 1 Properties of Paramagnetic Nanoparticles. Gd-DTPA-BOA Gd-DTPA-PEParticle Size (nm) 287 261 Polydispersity Index 0.28 0.23 [Gd³⁺] (mM)3.36 5.79 Gd³⁺ Ions/Particle 56,900 73,600 [Particles] (nM) 59.1 78.7

The particles were diluted to 0, 4, 6, 8, 10 and 12% PFOB (v/v) withdistilled deionized water. The initial nanoparticle formulationcontained 26.1 mol/L ¹⁹ F and the diluted aliquots had 0, 3.915, 5.22,6.525 and 7.83 mol/L ¹⁹ F, respectively. Total gadolinium content wasdetermined by neutron activation analysis. The gadolinium contents ofthe Gd-DTPA-BOA nanoparticle dilutions were 0; 0.336; 0.504; 0.672;0.84; and 1.01 mmol/L Gd³⁺. The paramagnetic ion concentrations inGd-DTPA-PE samples were 0; 0.579; 0.869; 1.16; 1.45; and 1.74 mmol/LGd³⁺.

The proton longitudinal and transverse relaxation rates (1/T₁ and 1/T₂,respectively) of each sample were measured at 40° C. on a Bruker MQ20Minispec NMR Analyzer with a field strength of 0.47 T. T₁ was measuredusing an inversion recovery sequence with 10 inversion delay values,while T₂ was measured with a Carr-Purcell-Meiboom-Gill (CPMG) sequence.The T₁ and T₂ relaxivities (i.e., ρ₁ and ρ₂, respectively) werecalculated from the slope of the linear least-squares regression oflongitudinal and transverse relaxation rates versus Gd³⁺ (i.e., ionrelaxivity) or nanoparticle (i.e., particle relaxivity) concentrationsand are reported in units of (s*mM)⁻¹.

Spin echo images from a clinical scanner (Gyroscan NT, PowerTrak 6000,Philips Medical Systems, Best, Netherlands) obtained with a standard 11cm diameter surface coil were used to measure the relaxivity of the twonanoparticle formulations at 1.5 T. A six chamber phantom allowed allsix dilutions to be studied in parallel. To accommodate the differentrelaxation times of the two paramagnetic formulations, different imagingparameters were applied. T₁ was calculated from an inversion recoveryMRI pulse sequence. The measurement for the Gd-DTPA-BOA phantom includedsix inversion times (T₁) ranging from 50 to 1500 ms, while theGd-DTPA-PE value utilized seven T₁ values ranging from 5 ms to 200 ms.The signal intensity (S1) from each chamber was fit to the equation:S1_(T1) =S1₀*(1-EXP(-T ₁ /T ₁)),   [1]where S1 ₀ represents the equilibrium signal intensity. The T₂ value forGd-DTPA-BOA was derived from a multi-echo sequence with 8 echo times(TE) ranging from 20 ms to 160 ms. Nine separate images with echo timesranging from 4.5 ms to 200 ms were used to calculate the T₂ relaxationfor the Gd-DTPA-PE phantom. MRI signal intensity was fit to theequation:S1_(TE) =S1₀*EXP(-TE/T ₂).   [2]The imaging parameters common for both formulations were: TR=1000 ms,TE=5 ms (unless otherwise noted), number of signal averages=4, imagematrix=128 by 128, FOV=7 cm by 7 cm, flip angle=90°, slice thickness=5mm.

The relaxivities of the two paramagnetic formulations were also measuredwith a 4.7 T magnet interfaced to a Varian INOVA console (VarianAssociates, Palo Alto, Calif.) using a 5 cm birdcage coil. As statedearlier, a six chamber phantom was used to study the various emulsiondilutions concurrently. T₁ and T₂ values were obtained with inversionrecovery (TE=7.2 ms, T₁ varied from 1 to 800 ms) and spin echo (TEvaried from 7.2 to 100 ms) pulse sequences, respectively. The imageswere collected with TR=3000 ms, number of signal averages 4, imagematrix=256 by 256, FOV=4 cm by 4 cm, flip angle=90°, slice thickness 2mm.

Finally, the relaxivities of the two paramagnetic preparations weremeasured independently at magnetic fields ranging from 0.05 T to 1.3 T(2-56 MHz) using a custom built variable field relaxometer (SouthwestResearch Institute, San Antonio, Tex.). The samples were measured attemperatures of 3° and 37° C. A saturation recovery pulse sequence with32 incremental τ values was used to measure ρ₁, while ρ₂ was measuredusing a CPMG pulse sequence with 500 echoes and a 2 ms inter-echo delaytime.

Table 2 shows T₁ and T₂ relaxivities of the Gd-DTPA-BOA and Gd-DTPA-PEparamagnetic formulations determined at three magnetic field strengths.

TABLE 2 Relaxivities of Gd-DTPA-BOA and Gd-DTPA-PE emulsions at threedifferent field strengths Ion-Based Relaxivity Particle-Based MagneticParamagnetic (s * mM)⁻¹ Relaxivity (s * mM)⁻¹ Field Chelate ρ₁ ρ₂ ρ₁ ρ₂0.47 T  Gd-DTPA-BOA 21.3 ± 0.2 23.8 ± 0.3 1,210,000 ± 10,000 1,350,000 ±20,000 Gd-DTPA-PE 36.9 ± 0.5 42.3 ± 0.6 2,710,000 ± 40,000 3,110,000 ±50,000 1.5 T Gd-DTPA-BOA 17.7 ± 0.2 25.3 ± 0.6 1,010,000 ± 10,0001,440,000 ± 30,000 Gd-DTPA-PE 33.7 ± 0.7  50 ± 2  2,480,000 ± 50,000 3,700,000 ± 100,000 4.7 T Gd-DTPA-BOA  9.7 ± 0.2 29.4 ± 0.3  549,000 ±9,000 1,670,000 ± 20,000 Gd-DTPA-PE 15.9 ± 0.1   80 ± 0.7 1,170,000 ±6,000  5,880,000 ± 50,000

At all magnetic field strengths, both the ion-based and particle-basedρ₁ of the Gd-DTPA-PE formulation were about two-fold greater (p<0.05)than ρ₁ of the Gd-DTPA-BOA agent. Similarly, ion-based andparticle-based ρ₂ of the Gd-DTPA-PE agent were approximately two-foldhigher (p<0.05) than ρ₂ of the Gd-DTPA-BOA system at the lowest magneticfield strength (0.47 T), and this relative difference was more thanthree-fold greater (p<0.05) at the highest field strength (4.7 T).

At 1.5 T, a typical medical imaging field strength, the ion-based ρ₁ andρ₂ for Gd-DTPA-BOA were 17.7±0.2 (s*mM)⁻¹ (mean±standard error) and25.3±0.6 (s*mM)⁻¹, respectively, consistent with our previous reportedestimates (Flacke, S., et al., Circulation (2001) 104:1280-1285).Incorporation of Gd-DTPA-PE (as opposed to Gd-DTPA-BOA) increased theion-based ρ₁ and ρ₂ to 33.7±0.7 (s*mM)⁻¹ and 50.0±2 (s*mM)⁻¹,respectively. More importantly from a targeted agent perspective, theparticle-based ρ₁ and ρ₂ for Gd-DTPA-BOA were 1,010,000±10,000 (s*mM)⁻¹and 1,440,000±30,000 (s*mM)⁻¹, respectively, and for Gd-DTPA-PEnanoparticles the particle-based ρ₁ and ρ₂ were 2,480,000±50,000(s*mM)⁻¹ and 3,700,000±100,000 (s*mM)⁻¹, respectively. To our knowledge,particulate or molecular relaxivities in these ranges are the highestvalues reported to date for any targeted or blood pool paramagneticcontrast agent at these field strengths.

Magnetic field strength influences relaxivity. The magnitudes of ion andparticle longitudinal relaxivities decline as magnetic field strengthincreased from 0.47 T to 4.7 T, whereas the ion and particle transverserelaxivities progressively increased with higher field strengths.Although the particle longitudinal relaxivity declined about 50% at 4.7T compared to 1.5 T, the particle ρ₁ remained very high. As aligand-targeted contrast agent, the decreases in relaxivity at higherfield strengths will be effectively offset by reduced voxel sizes,smaller partial volume dilution effects and improved signal to noise.

Variable field relaxometry measurements show that ρ₁ of both emulsionswas dominated by the long correlation time (τ_(c)) of the slowlytumbling emulsion complex (FIG. 3). In fact, the particles wererelatively so large, that there was almost no field dependence(dispersion). In contrast, the ρ₂ values initially followed those of ρ₁but did not decrease at higher fields in accordance with expectationsbased on the Solomon-Bloembergen equations (Wood, M. L., J. Mag. Res.Imag. (1993) 3:149-156) (due to the non-dispersive term involvingτ_(c)). For the Gd-DTPA-BOA emulsion, the “peak” ρ₁ relaxivity wasaround 25 (s*mM)⁻¹ and the maximum value of ρ₂ was 30 (s*mM)⁻¹. Thevalue of ρ₁ was largely independent of temperature, but ρ₂ increased atthe lower temperature. For the Gd-DTPA-PE emulsion, however, therelaxivities were much higher, with ρ₁ reaching 40 (s*mM)⁻¹ at 40 MHz(approx. 1.7 T) and ρ₂ reaching 50 (s*mM)⁻¹ at 56 MHz (1.3 T). Thetemperature dependence of Gd-DTPA-PE was also different from Gd-DTPA-BOAwith ρ₁ decreasing at the lower temperature and ρ₂ remaining independentof temperature. The relaxometry values were consistent with analogousmeasurements made at 0.47 T and 1.5 T (Table 2). Moreover, thetemperature dependence of these curves suggested that the Gd-DTPA-PEchelate has better access to water (i.e., faster exchange) compared toGd-DTPA-BOA.

EXAMPLE 5 Enhanced Relaxivity of Contrast Agent Coupled to Nanoparticles

The Gd-MeO-DOTA-PE and Gd-MeO-DOTA-triglycine-PE conjugates wereassociated with nanoparticles prepared as in Preparation A andassociated with the nanoparticles as described in that Preparation. Eachparticle contains approximately 33,000 Gd³⁺ chelates. The ρ₁ relaxivitywas compared, as described in Example 4, with the relaxivities obtainedfrom similar nanoparticles coupled with similar amounts of Gd-DTPA-BOAand Gd-DTPA-PE. The results are shown on a per ion basis and perparticle basis in FIGS. 4 and 5, respectively.

The ρ₁ value on a per ion basis for Gd-DTPA-BOA nanoparticles was 21.3s*mM⁻¹; that of Gd-MeO-DOTA-PE nanoparticles is 29.8 s*mM⁻¹, and ofGd-MeO-DOTA-triglycine-PE nanoparticles is 33.0 s*mM⁻¹. Since eachparticle carries 33,000 Gd³⁺-chelates, the particulate relaxivities wereGd-DTPA-BOA: 700,000 s*mM⁻¹, Gd-MeO-DOTA-PE: 980,000 s*mM⁻¹ andGd-MeO-DOTA-Triglycine-PE: 1,100,000 s*mM⁻¹. It is seen that thetriglycine spacer improves relaxivity, and that the relaxivity for bothconjugates using DOTA and PE spacer are improved over that ofGd-DTPA-BOA.

EXAMPLE 6 Transmetalation

As described above, the coupled nanoparticles may be coupled to atargeting agent which will delay clearance from the subject as comparedto non-targeted chelating agents such as blood pool contrast agents.This makes retention of metal by the chelate of significance; retentionof metal by macrocyclic chelates, such as DOTA is known to be orders ofmagnitude stronger than for linear chelates such as DTPA or EDTA.Further, use of a coupling site not a part of the chelator itselfresults in efficient coupling without sacrificing chelation strength.

An excess of zinc was used as a competing species to producetransmetalation in GD-DTPA-BOA nanoparticles and nanoparticles coupledto the invention conjugates. The longitudinal relaxation of Gd-DTPA-BOAnanoparticles decreased quickly after addition of ZnCl₂, whereas neitherDOTA chelate showed a high rate or high magnitude of change inrelaxivity, reflecting the improved stability of the inventionGd³⁺complexes. The retained gadolinium at equilibrium was much higherfor the two DOTA chelates (91%) compared to the DTPA chelate (75%).Thus, the DOTA chelates demonstrated 40-55% higher relaxivity and 64%lower transmetalation than the linear Gd-DTPA-BOA chelate.

EXAMPLE 7 Effect of Cleavable Linker on Clearance

Gd-MeO-DOTA-PE and Gd-MeO-DOTA-triglycine-PE nanoparticles wereadministered intravenously to Sprague Dawley rats at standard dosages,i.e., 0.5 ml/kg of 40 wt % perfluorocarbon emulsion. Livers and spleenswere obtained from each animal (n=3 per treatment) at each time pointand analyzed for gadolinium. The results are shown as percent ofinjected dose retained per organ in FIGS. 6A (liver) and 6B (spleen). Asshown, the formulation containing the cleavable triglycine linker ismore rapidly cleared.

EXAMPLE 8 Tumor Imaging

A. DSPE-PEG(2000)Maleimide-Mercaptoacetic Acid Adduct

1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Maleimide(PolyethyleneGlycol)2000] is dissolved in DMF and degassed by sparging with nitrogenor argon. The oxygen-free solution is adjusted to pH 7-8 using DIEA, andtreated with mercaptoacetic acid. Stirring is continued at ambienttemperatures until analysis indicates complete consumption of startingmaterials. The solution is used directly in the following reaction.

B. Conjugation of the DSPE-PEG(2000)Maleimide-Mercaptoacetic Acid AdductWith Anti-Tumor Associated Antigen

The product solution of Part A, above, is pre-activated by the additionof HBTU and sufficient DIEA to maintain pH 8-9. To the solution is addedmonoclonal antibody specific for tumor associated antigen, and thesolution is stirred at room temperature under nitrogen for 18 h. DMF isremoved in vacuo and the crude product is purified by preparative HPLCto obtain the PE coupled through a linker to anti-tumor antibodies.

C. Preparation of Nanoparticles:

The paramagnetic nanoparticles are produced as described in Flacke, S.,et al., Circulation (2001) 104:1280-1285. Briefly, the nanoparticulateemulsions are comprised of 40% (v/v) perfluorooctylbromide (PFOB), 2%(w/v) of a surfactant co-mixture, 1.7% (w/v) glycerin and waterrepresenting the balance.

The surfactant of control, i.e., non-targeted, paramagnetic emulsionsincluded 60 mole% lecithin (Avanti Polar Lipids, Inc., Alabaster, Ala.),8 mole % cholesterol (Sigma Chemical Co., St. Louis, Mo.), 2 mole %dipalmitoyl-phosphatidylethanolamine (DPPE) (Avanti Polar Lipids, Inc.,Alabaster, Ala.) and 30 mole % gadolinium diethylenetriaminepentaaceticacid-bisoleate (Gd-DTPA-BOA, Gateway Chemical Technologies, St. Louis,Mo.). The preparation of chelate is described in Example 1.

Tumor-targeted paramagnetic nanoparticles are prepared as above with asurfactant co-mixture that included: 60 mole % lecithin, 0.05 mole % ofthe conjugate of paragraph B, 8 mole % cholesterol, 30 mole % Example 1chelate containing Gd³⁺ and 1.95 mole % DPPE.

Tumor-targeted non-paramagnetic nanoparticles are prepared in anidentical fashion to the targeted formulation excluding the addition ofthe lipophilic Gd³⁺ chelate, which is substituted in the surfactantco-mixture with increased lecithin (70 mole %) and cholesterol (28 mole%).

The components for each nanoparticle formulation are emulsified in aM110S Microfluidics emulsifier (Microfluidics, Newton, Mass.) at 20,000PSI for four minutes. The completed emulsions are placed in crimp-sealedvials and blanketed with nitrogen.

D. Tumor Model

Male New Zealand White Rabbits (˜2.0 kg) are anesthetized withintramuscular ketamine and xylazine (65 and 13 mg/kg, respectively). Theleft hind leg of each animal is shaved, sterile prepped and infiltratedlocally with Marcaine™ prior to placement of a small incision above thepopliteal fossa. A 2 by 2 by 2 mm³ Vx-2 carcinoma tumor fragment,freshly obtained from a donor animal, is implanted at a depth ofapproximately 0.5 cm. Anatomical planes are reapproximated and securedwith a single absorbable suture. Finally, the skin incision is sealedwith Dermabond skin glue. Following the tumor implantation procedure,the effects of xylazine are reversed with yohimbine and animals areallowed to recover.

Twelve days after Vx-2 implantation rabbits are anesthetized with 1% to2% Isoflurane™, intubated, ventilated and positioned within the bore ofthe MRI scanner for study. Intravenous and intraarterial catheters,placed in opposite ears of each rabbit, are used for systemic injectionof nanoparticles and arterial blood sampling as described below. Animalsare monitored physiologically throughout the study in accordance with aprotocol and procedures approved by the Animal Studies Committee atWashington University Medical School.

At 12 days post-implantation, Vx-2 tumor volumes of animals receivingtumor-targeted (130±39 mm³) or non-targeted nanoparticles (148±36 mm³)were not different (p>0.05).

Twelve New Zealand rabbits implanted with Vx-2 tumors, as describedabove, are randomized into three treatment regimens and received either:

-   -   1) tumor-targeted paramagnetic nanoparticles (tumor-targeted,        n=4),    -   2) non-targeted paramagnetic nanoparticles (i.e., control group,        n=4), or    -   3) tumor-targeted non-paramagnetic nanoparticles followed by        tumor-targeted paramagnetic nanoparticles (i.e., competition        group, n=4).

In treatment groups 1 and 2, rabbits receive 0.5 ml/kg of tumor-targetedor control paramagnetic nanoparticles following the acquisition ofbaseline MR images. In treatment group 3, all rabbits receive 0.5 ml/kgtumor-targeted non-paramagnetic nanoparticles two hours before MRimaging followed by 0.5 ml/kg tumor-targeted paramagnetic nanoparticles.Dynamic MR images are obtained at injection and every 30 minutes foreach animal over two hours to monitor initial changes in signalenhancement in the tumor and muscle regions. All tumors are resected andfrozen for histology to corroborate MR molecular imaging results.

E. Magnetic Resonance Imaging and Histology Procedures

Twelve days after tumor implantation, the animals undergo MRI scanningon a 1.5 Tesla clinical scanner (NT Intera with Master Gradients,Philips Medical Systems, Best, Netherlands). Each animal is placedinside a quadrature head/neck birdcage coil with an 11 cm diametercircular surface coil positioned against the hindlimb near the tumor.The quadrature body coil is used for all radio-frequency transmission;the birdcage coil is used for detection during scout imaging; and thesurface coil is used for detection during high-resolution imaging. A 10ml syringe filled with gadolinium diethylenetriaminepentaacetic acid(Gd-DTPA) doped water is placed within the high-resolution field of view(FOV) and served as a signal intensity standard.

Tumors are initially localized at the site of implantation with aT₂-weighted turbo spin-echo scan (TR: 2000 ms, TE: 100 ms, FOV: 150 mm,slice thickness: 3 mm, matrix: 128 by 256, signal averages: 2, turbofactor: 3, scan time: 3 min). A high-resolution, T₁ -weighted, fatsuppressed, three-dimensional, gradient echo scan (TR: 40 ms, TE: 5.6ms, FOV: 64 mm, slice thickness: 0.5 mm, contiguous slices: 30, in-planeresolution: 250 μm, signal averages: 2, flip angle: 650, scan time: 15min) of the tumor is collected at baseline and repeated immediately and30, 60, 90 and 120 minutes after paramagnetic nanoparticle injection.

Tumor volumes are calculated on an offline image processing workstation(EasyVision v5.1, Philips Medical Systems, Best, Netherlands).Regions-of-interest (ROI) were applied manually around the tumor in eachslice of the T₁-weighted baseline scan, are combined into athree-dimensional object and the volume calculated.

To quantify image enhancement over time, an unbiased image analysisprogram is used. T₁-weighted images (three contiguous slices through thecenter of each tumor) collected before, immediately after and 30, 60, 90and 120 minutes after intravenous nanoparticle injection are analyzedwith MATLAB (The MathWorks, Inc., Natick, Mass.). The image intensity ateach timepoint is normalized to the baseline image via the referencegadolinium standard. Serial images are spatially co-registered andcontrast enhancement is determined for each pixel at each post-injectiontimepoint. An ROI is manually drawn around a portion of the hindlimbmuscle in the baseline images and the average pixel-by-pixel signalenhancement inside the ROI is calculated at each timepoint. A second ROIis manually drawn around the tumor and the standard deviation of thetumor signal is calculated in the baseline image for each animal. Pixelsare considered enhanced when signal intensity is increased by greaterthan three times the standard deviation of the tumor signal at baseline(i.e., enhancement greater than 99% of the variation seen at baseline).Solitary enhancing pixels, those in which all surrounding in-planepixels do not enhance, are removed from the calculations as noise. Theremaining enhancing pixel clusters are mapped back to the immediate, 30,60 and 90 minute images and the average signal increase at each intervalis determined. Statistical comparisons are performed for tumor andmuscle for each timepoint using ANOVA (SAS, SAS Institute, Cary, N.C.).Treatment means are separated using the LSD procedure (p<0.05).

After imaging, tumors are resected for histology andimmunohistochemistry to verify tumor pathology and assess associatedvascularity and angiogenesis. Tumors are frozen (−78° C.) in OCT mediumwith known orientation relative to original anatomical position and theMRI image planes. Four micron frozen sections (Leica Microsystems, Inc.,Bannockburn, Ill.), fixed in acetone at −20° C. for 15 minutes and airdried overnight (4° C.), are stained with hematoxylin-eosin , murineanti-human/rabbit endothelium antibody (QBEND/40, 1:10 dilution,Research Diagnostics, Inc., Flanders, N.J.), or a murine anti-humanα_(ν)β₃-integrin (LM-609, 1:200 dilution, Chemicon International,Temecula, Calif.). Immunohistochemistry is performed using theVectastain ® Elite ABC kit (Vector Laboratories, Burlingame, Calif.94010), developed with the Vector® VIP kit, counterstained with Vector®methylgreen nuclear counterstain. Slides are reviewed with a NikonEclipse E800 research microscope (Nikon USA, Melville, N.Y.) equippedwith a Nikon digital camera (Model DXM 1200) and captured with NikonACT-1 software.

1. A compound of the formula:

wherein Ch represents a chelating moiety selected from the groupconsisting of porphyrins, ethylenediaminetetraacetic acid (EDTA),diethylenetriamine-N,N,N′,N″,N″-pentaacetate (DTPA),1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7 (ODDA),16-diacetate,N-2-(azol-1(2)-yl)ethyliminodiacetic acids,1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),1,7,13-triaza-4,10,16-trioxacyclo-octadecane-N,N′,N′″-triacetate (TTTA),tetraethylene glycols,1,5,9-triazacyclododecane-N,N′,N″-tris(methylenephosphonic acid (DOTRP),and N,N′,N″-trimethylammonium chloride (DOTMA); m is 0-3; each R¹ isindependently a non-interfering substituent selected from the groupconsisting of halo, OR, NR₂, SR, CN, NO₂, SO₃H, and R where R is alkylor alkenyl optionally substituted by halo, or ═O, and optionallycontaining a heteroatom, such as O, S or N; 1 is 0-2; Z is S or O; R² isH or alkyl (1-4C); n is 1; and each R³ is independently an optionallysubstituted saturated or unsaturated hydrocarbyl group containing atleast 10 C; and spacer is —CH₂CH₂— and/or includes a peptide, apseudopeptide, or a polyalkylene glycol, optionally containing acleavage site.
 2. The compound of claim 1, wherein the spacer is CH₂CH₂and R² is H.
 3. The compound of claim 1, wherein R² is H, 1 is 0 or 1and m is 1 or
 0. 4. The compound of claim 1, wherein each R³COO is aresidue of a naturally occurring fatty acid or a mixture of saidresidues.
 5. The compound of claim 1, wherein R¹ is CH₃O.
 6. Thecompound of claim 1, wherein the spacer is a peptide or a polyalkyleneglycol.
 7. The compound of claim 1, which further comprises, associatedwith Ch, a paramagnetic metal.
 8. A composition which comprises thecompound of claim 7 associated with lipophilic nanoparticles ormicroparticles, and wherein said particles contain at least 2,000 copiesof said compound.
 9. The composition of claim 8, wherein thenanoparticles or microparticles further contain a targeting agent. 10.The composition of claim 8, wherein said microparticles or nanoparticlesfurther comprise a biologically active agent.
 11. The composition ofclaim 8, wherein said microparticles or nanoparticles are liposomes, oildroplets, perfluorocarbon nanoparticles, lipid-coated protein particles,or lipid-coated polysaccharides.
 12. A method to obtain a magneticresonance image which method comprises imaging a tissue which isassociated with the composition of claim
 9. 13. The compound of claim 7,wherein the paramagnetic metal ion is Gd(III), Mn(II), iron, europiumand/or dysprosium.
 14. The compound of claim 13, wherein theparamagnetic metal ion is Gd(III).
 15. A composition which comprises thecompound of claim 13 associated with lipophilic nanoparticles ormicroparticles, and wherein said particles contain at least 2,000 copiesof said compound.
 16. The composition of claim 15, wherein thenanoparticles or microparticles further contain a targeting agent.
 17. Amethod to obtain a magnetic resonance image which method comprisesimaging a tissue which is associated with the composition of claim 16.18. A composition which comprises the compound of claim 14 associatedwith lipophilic nanoparticles or microparticles, and wherein saidparticles contain at least 2,000 copies of said compound.
 19. Thecomposition of claim 18, wherein the nanoparticles or microparticlesfurther contain a targeting agent.
 20. A method to obtain a magneticresonance image which method comprises imaging a tissue which isassociated with the composition of claim
 19. 21. A compound of theformula:

wherein Ch represents a chelating moiety; m is 0-3; each R¹ isindependently selected from the group consisting of halo, OR, NR₂, SR,CN, NO₂, SO₃H, and R where R is alkyl or alkenyl optionally substitutedby halo, or ═O, and optionally containing a heteroatom, such as O, S orN; 1 is 0-2; Z is S or O; R² is H or alkyl (1-4C); n is 1; and each R³is independently an optionally substituted saturated or unsaturatedhydrocarbyl group containing at least 10C; and spacer is —CH₂CH₂— and/orincludes a peptide, a pseudopeptide, or a polyalkylene glycol,optionally containing a cleavage site; which further comprises,associated with CH, a paramagnetic metal ion.
 22. A composition whichcomprises the compound of claim 21 associated with lipophilicnanoparticles or microparticles, and wherein said particles contain atleast 2,000 copies of said compound.
 23. The composition of claim 22,wherein the nanoparticles or microparticles further contain a targetingagent.
 24. The composition of claim 22, wherein said microparticles ornanoparticles further comprise a biologically active agent.
 25. Thecomposition of claim 22, wherein said microparticles or nanoparticlesare liposomes, oil droplets, perfluorocarbon nanoparticles, lipid-coatedprotein particles, or lipid-coated polysaccharides.
 26. A method toobtain a magnetic resonance image which method comprises imaging atissue which is associated with the composition of claim
 23. 27. Thecompound of claim 21, wherein the paramagnetic metal ion is Gd(III),Mn(II), iron, europium and/or dysprosium.
 28. The compound of claim 27,wherein the paramagnetic metal ion is Gd(III).